Prognostic and predictive impact of metastatic organ involvement on maintenance therapy in advanced metastatic colorectal cancer: Subgroup analysis of patients treated within the PanaMa trial (AIO KRK 0212).

Despite molecular selection, patients (pts) with RAS wildtype mCRC represent a heterogeneous population including diversity in metastatic spread. We investigated metastatic patterns for their prognostic and predictive impact on maintenance therapy with 5-fluorouracil/folinic acid ± panitumumab. The study population was stratified according to (1) number of involved metastatic sites (single vs multiple organ metastasis), liver-limited disease vs (2) liver metastasis plus one additional site, and (3) vs liver metastasis plus ≥two additional sites. Kaplan-Meier method and Cox regressions were used to correlate efficacy endpoints. Single organ metastasis was observed in 133 pts (53.6%) with 102 pts (41.1%) presenting with liver-limited disease, while multiple organ metastases were reported in 114 pts (46.0). Multiple compared to single organ metastases were associated with less favorable PFS (HR 1.48, 95% CI 1.13-1.93; P = .004) and OS (HR 1.37, 95% CI 0.98-1.93; P = .068) of maintenance therapy. While metastatic spread involving one additional extrahepatic site was not associated with clearly impaired survival compared to liver-limited disease, pts with liver metastasis plus ≥two additional sites demonstrated less favorable PFS (HR 1.92, 95% CI 1.30-2.83; P < .001), and OS (HR 2.38, 95% CI 1.51-3.76; P < .001) of maintenance therapy. Pmab-containing maintenance therapy appeared active in both pts with multiple (HR 0.58; 95% CI, 0.39-0.86; P = .006) as well as to a lesser numerical extent in pts with single organ metastasis (HR 0.83; 95% CI, 0.57-1.21; P = .332; Interaction P = .183). These data may support clinical decisions when EGFR-based maintenance therapy is considered.

Antithetical NFATc1-Sox2 and p53-miR200 signaling networks govern pancreatic cancer cell plasticity.

In adaptation to oncogenic signals, pancreatic ductal adenocarcinoma (PDAC) cells undergo epithelial-mesenchymal transition (EMT), a process combining tumor cell dedifferentiation with acquisition of stemness features. However, the mechanisms linking oncogene-induced signaling pathways with EMT and stemness remain largely elusive. Here, we uncover the inflammation-induced transcription factor NFATc1 as a central regulator of pancreatic cancer cell plasticity. In particular, we show that NFATc1 drives EMT reprogramming and maintains pancreatic cancer cells in a stem cell-like state through Sox2-dependent transcription of EMT and stemness factors. Intriguingly, NFATc1-Sox2 complex-mediated PDAC dedifferentiation and progression is opposed by antithetical p53-miR200c signaling, and inactivation of the tumor suppressor pathway is essential for tumor dedifferentiation and dissemination both in genetically engineered mouse models (GEMM) and human PDAC. Based on these findings, we propose the existence of a hierarchical signaling network regulating PDAC cell plasticity and suggest that the molecular decision between epithelial cell preservation and conversion into a dedifferentiated cancer stem cell-like phenotype depends on opposing levels of p53 and NFATc1 signaling activities.

Context-Dependent Epigenetic Regulation of Nuclear Factor of Activated T Cells 1 in Pancreatic Plasticity.

BACKGROUND & AIMS: The ability of exocrine pancreatic cells to change the cellular phenotype is required for tissue regeneration upon injury, but also contributes to their malignant transformation and tumor progression. We investigated context-dependent signaling and transcription mechanisms that determine pancreatic cell fate decisions toward regeneration and malignancy. In particular, we studied the function and regulation of the inflammatory transcription factor nuclear factor of activated T cells 1 (NFATC1) in pancreatic cell plasticity and tissue adaptation. METHODS: We analyzed cell plasticity during pancreatic regeneration and transformation in mice with pancreas-specific expression of a constitutively active form of NFATC1, or depletion of enhancer of zeste 2 homologue 2 (EZH2), in the context of wild-type or constitutively activate Kras, respectively. Acute and chronic pancreatitis were induced by intraperitoneal injection of caerulein. EZH2-dependent regulation of NFATC1 expression was studied in mouse in human pancreatic tissue and cells by immunohistochemistry, immunoblotting, and quantitative reverse transcription polymerase chain reaction. We used genetic and pharmacologic approaches of EZH2 and NFATC1 inhibition to study the consequences of pathway disruption on pancreatic morphology and function. Epigenetic modifications on the NFATC1 gene were investigated by chromatin immunoprecipitation assays. RESULTS: NFATC1 was rapidly and transiently induced in early adaptation to acinar cell injury in human samples and in mice, where it promoted acinar cell transdifferentiation and blocked proliferation of metaplastic pancreatic cells. However, in late stages of regeneration, Nfatc1 was epigenetically silenced by EZH2-dependent histone methylation, to enable acinar cell redifferentiation and prevent organ atrophy and exocrine insufficiency. In contrast, oncogenic activation of KRAS signaling in pancreatic ductal adenocarcinoma cells reversed the EZH2-dependent effects on the NFATC1 gene and was required for EZH2-mediated transcriptional activation of NFATC1. CONCLUSIONS: In studies of human and mouse pancreatic cells and tissue, we identified context-specific epigenetic regulation of NFATc1 activity as an important mechanism of pancreatic cell plasticity. Inhibitors of EZH2 might therefore interfere with oncogenic activity of NFATC1 and be used in treatment of pancreatic ductal adenocarcinoma.

Evaluation of an interdisciplinary palliative care inhouse training for professionals in gynecological oncology.

PURPOSE: The aim of this study was to evaluate the effect of a pilot interdisciplinary inhouse training in palliative care (PC) for gynecological oncologists. METHODS: Competencies of participants from a gynecological university department were evaluated taking part in an interdisciplinary PC course in a pre and post design. The multiprofessional course covered basic principles of PC, symptom management and communication taught by PC specialists. Competencies were evaluated using self-designed questionnaires before (ISPG-1), right after (ISPG-2), and 6 months after the training (ISPG-3) (inhouse seminar palliative care in gynecology: ISPG). RESULTS: 31 persons from the department of gynecology took part in the course, of which 27 answered the first questionnaire (seven nurses (26%), 19 doctors (71%), one profession not indicated (3%), median working experience in gynecological oncology: 5 years). Return rates were: ISPG-1 27/31 (87.1%), ISPG-2 20/31 (64.5%) and IPSG-3 14/31 (45.2%). A more positive attitude towards PC could be observed in the majority of participants after the course (ISPG-2 62%, ISPG-3 71%). They felt more competent in the care of palliative patients (46%). PC would be initiated earlier and the interaction with other disciplines was improved (ISPG-2 85%, ISPG-3 100%). The participants assessed a significant improvement of their skills in all palliative fields which were analyzed. CONCLUSION: PC inhouse training improves the understanding of PC and the interdisciplinary approach in the management of patients with advanced disease. It is a feasible and useful instrument to improve the competencies in generalist PC of specialists in gynecological oncology.

The utilization of multidisciplinary tumor boards (MDT) in clinical routine: results of a health care research study focusing on patients with metastasized colorectal cancer.

PURPOSE: Multidisciplinary tumor boards (MDT) have been advocated as standard of care in modern oncology. German guidelines for metastasized colorectal cancer (mCRC) recommend MDT discussion of colon cancer patients after completion of primary tumor therapy but stage IV colon cancer as well as rectal cancer patients prior to any therapy. In this health care research study, we evaluated application and decisional consequences of this approach in clinical routine. METHODS: All major institutions providing oncological care in southern Lower Saxony and Northern Hesse (N = 11) were invited. Patients with mCRC diagnosed between 01/2011 and 12/2013 were eligible. Data were collected using a standardized patient report form and stored in a GCP-conform EDC-system (secuTrial®). RESULTS: A university medical center, four teaching hospitals, one communal hospital, and three oncological focus practices participated in the study. In total, 470 patients with a median age of 70 years were enrolled. Guideline conform MDT discussion was performed in 63% of operated colon cancer patients, 38% of stage IV colon cancer patients and 47% of rectal cancer patients, respectively. Resection of metastases was performed in 41% of cases. Patients ≥70 years (n = 250) received significantly more often treatment following MDT discussion (86 versus 64%, p = 0.0002). Not the resection rate (48 versus 57%, p = 0.1574) but indication for preoperative chemotherapy (57 versus 33%, p = 0.0056) significantly differed when patients with single organ metastases experienced MDT discussion. CONCLUSIONS: MDT discussion is not as established as advocated by national guidelines. Treatment decisions differ especially in older patients and those with single organ metastases.

NFATc1 Links EGFR Signaling to Induction of Sox9 Transcription and Acinar-Ductal Transdifferentiation in the Pancreas.

BACKGROUND & AIMS: Oncogenic mutations in KRAS contribute to the development of pancreatic ductal adenocarcinoma, but are not sufficient to initiate carcinogenesis. Secondary events, such as inflammation-induced signaling via the epidermal growth factor receptor (EGFR) and expression of the SOX9 gene, are required for tumor formation. Herein we sought to identify the mechanisms that link EGFR signaling with activation of SOX9 during acinar-ductal metaplasia, a transdifferentiation process that precedes pancreatic carcinogenesis. METHODS: We analyzed pancreatic tissues from Kras(G12D);pdx1-Cre and Kras(G12D);NFATc1(Δ/Δ);pdx1-Cre mice after intraperitoneal administration of caerulein, vs cyclosporin A or dimethyl sulfoxide (controls). Induction of EGFR signaling and its effects on the expression of Nuclear factor of activated T cells c1 (NFATc1) or SOX9 were investigated by quantitative reverse-transcription polymerase chain reaction, immunoblot, and immunohistochemical analyses of mouse and human tissues and acinar cell explants. Interactions between NFATc1 and partner proteins and effects on DNA binding or chromatin modifications were studied using co-immunoprecipitation and chromatin immunoprecipitation assays in acinar cell explants and mouse tissue. RESULTS: EGFR activation induced expression of NFATc1 in metaplastic areas from patients with chronic pancreatitis and in pancreatic tissue from Kras(G12D) mice. EGFR signaling also promoted formation of a complex between NFATc1 and C-JUN in dedifferentiating mouse acinar cells, leading to activation of Sox9 transcription and induction of acinar-ductal metaplasia. Pharmacologic inhibition of NFATc1 or disruption of the Nfatc1 gene inhibited EGFR-mediated induction of Sox9 transcription and blocked acinar-ductal transdifferentiation and pancreatic cancer initiation in mice. CONCLUSIONS: EGFR signaling induces expression of NFATc1 and Sox9, leading to acinar cell transdifferentiation and initiation of pancreatic cancer. Strategies designed to disrupt this pathway might be developed to prevent pancreatic cancer initiation in high-risk patients with chronic pancreatitis.

Management of a metastasized high grade insulinoma (G3) with refractory hypoglycemia: case report and review of the literature.

Insulinomas represent the most common functional neuroendocrine tumor of the pancreas. They are usually solitary, benign, well differentiated (G1/G2) and curable by surgery. We describe the case of a 45 year old male Caucasian with a unique malignant, metastasized pancreatic insulinoma (Ki 67 of 70%, G3). To control excessive insulin production emanating in refractory hypoglycemia and growth of the highly proliferating tumor a multimodal therapeutic approach including the consecutive use of tumor debulking surgery, chemotherapy, TACE, SIRT, PRRT as well as a drug therapy with diazoxide, somatostatin analogs and everolimus was employed. Chemotherapy with carboplatin/etoposide plus everolimus provided the longest normoglycemic period. After progress chemotherapy with dacarbazine had the most positive effect, while debulking approaches such as surgery and liver directed therapies, as well as PRRT were less efficient with only transient success.

The cytoskeletal adaptor protein IQGAP1 regulates TCR-mediated signaling and filamentous actin dynamics.

The Ras GTPase-activating-like protein IQGAP1 is a multimodular scaffold that controls signaling and cytoskeletal regulation in fibroblasts and epithelial cells. However, the functional role of IQGAP1 in T cell development, activation, and cytoskeletal regulation has not been investigated. In this study, we show that IQGAP1 is dispensable for thymocyte development as well as microtubule organizing center polarization and cytolytic function in CD8(+) T cells. However, IQGAP1-deficient CD8(+) T cells as well as Jurkat T cells suppressed for IQGAP1 were hyperresponsive, displaying increased IL-2 and IFN-γ production, heightened LCK activation, and augmented global phosphorylation kinetics after TCR ligation. In addition, IQGAP1-deficient T cells exhibited increased TCR-mediated F-actin assembly and amplified F-actin velocities during spreading. Moreover, we found that discrete regions of IQGAP1 regulated cellular activation and F-actin accumulation. Taken together, our data suggest that IQGAP1 acts as a dual negative regulator in T cells, limiting both TCR-mediated activation kinetics and F-actin dynamics via distinct mechanisms.

The effect of arm weight support on upper limb muscle synergies during reaching movements.

BACKGROUND: Compensating for the effect of gravity by providing arm-weight support (WS) is a technique often utilized in the rehabilitation of patients with neurological conditions such as stroke to facilitate the performance of arm movements during therapy. Although it has been shown that, in healthy subjects as well as in stroke survivors, the use of arm WS during the performance of reaching movements leads to a general reduction, as expected, in the level of activation of upper limb muscles, the effects of different levels of WS on the characteristics of the kinematics of motion and of the activity of upper limb muscles have not been thoroughly investigated before. METHODS: In this study, we systematically assessed the characteristics of the kinematics of motion and of the activity of 14 upper limb muscles in a group of 9 healthy subjects who performed 3-D arm reaching movements while provided with different levels of arm WS. We studied the hand trajectory and the trunk, shoulder, and elbow joint angular displacement trajectories for different levels of arm WS. Besides, we analyzed the amplitude of the surface electromyographic (EMG) data collected from upper limb muscles and investigated patterns of coordination via the analysis of muscle synergies. RESULTS: The characteristics of the kinematics of motion varied across WS conditions but did not show distinct trends with the level of arm WS. The level of activation of upper limb muscles generally decreased, as expected, with the increase in arm WS. The same eight muscle synergies were identified in all WS conditions. Their level of activation depended on the provided level of arm WS. CONCLUSIONS: The analysis of muscle synergies allowed us to identify a modular organization underlying the generation of arm reaching movements that appears to be invariant to the level of arm WS. The results of this study provide a normative dataset for the assessment of the effects of the level of arm WS on muscle synergies in stroke survivors and other patients who could benefit from upper limb rehabilitation with arm WS.

Guest editorial: From neuroscience to neuro-rehabilitation: transferring basic neuroscientific principles from laboratory to bedside.

Several new approaches for treatment of Central Nervous System (CNS) disorders are currently under investigation, including the use of rehabilitation training strategies, which are often combined with electrical and/or pharmacological modulation of spinal locomotor circuitries. While these approaches show great promise in the laboratory setting, there still exists a large gap in knowledge on how to transfer these treatments to daily clinical use. This thematic series presents a cross section of cutting edge approaches with the goal of transferring basic neuroscience principles from the laboratory to the proverbial "bedside".

Virtual reality aided training of combined arm and leg movements of children with CP.

Cerebral palsy (CP) occurs in over 2 out of 1000 live births and can impair motor control and cognition. Our goal was to create a robotic rehabilitation environment that mimics real-life situations by allowing simultaneous exercise of upper and lower limbs. We chose to use the Lokomat as a gait robot and added a novel removable arm robot, called PASCAL (pediatric arm support robot for combined arm and leg training), that was integrated into the Lokomat environment. We also added a virtual reality (VR) environment that enables the subject to perform motivating game-like scenarios incorporating combined arm and leg movements. In this paper we summarize the design of PASCAL and present the novel virtual environment including first experimental results. The next step will be to test whether a combined application of the virtual environment and the two simultaneously working robots is feasible in healthy participants, and finally to clinically evaluate the entire system on children with CP.

Delayed perforation of an atrial pacemaker electrode: Lifelong risk for a rare but serious complication.

Key Clinical Message: Nonspecific symptoms such as pleuritic or pericardial chest pain in cardiovascular implantable electronic devices patients, even with unremarkable ECG or device parameters, should always raise suspicion of electrode perforation, regardless of how long ago the implantation was performed. Abstract: We report the successful percutaneous management of a 77-year-old woman who had a dual-chamber pacemaker implanted more than 1 year ago and presented with pericarditis pain and compensated pericardial hemorrhagic tamponade. The symptoms were due to very late acute perforation of the atrial lead. This report is intended to raise awareness of procedure-related complications in the large group of cardiovascular implantable electronic device patients. Pleuritic or pericardial pain in these patients should raise suspicion of electrode perforation, as the risk of perforation is not restricted to the period immediately after implantation and a lifelong risk cannot apparently be excluded.

Biologically Based Complementary and Alternative Medicine Use in Breast Cancer Patients and Possible Drug-Drug Interactions.

Purpose: Biologically based complementary and alternative medicine (BB-CAM) is gaining importance. Cancer patients in particular are at risk of interactions between the prescribed medications (intravenous or oral anticancer therapy, concomitant medication, medication for pre-existing illnesses) and BB-CAM. This investigation aims to identify potentially clinically relevant interactions between both BB-CAM and conventional medicine and two BB-CAM products in breast cancer patients (n = 47). Methods: From March 2020 to January 2021, consecutive breast cancer patients (n = 47) completed a questionnaire about their medication and BB-CAM intake at the beginning of a new intravenous or oral tumor therapy (time point 1) and again after 10 to 12 weeks (time point 2) at the LMU Breast Center in Munich. The collective was divided into two subgroups based on the time after initial diagnosis; a cutoff of 6 months was used. The survey was available through an eHealth application called CANKADO as electronic patient-reported outcome only. Lexicomp® and AiD Klinik® databases were used for evaluating potentially clinically relevant interactions. As part of routine care, the collected data were evaluated and cross-checked in interdisciplinary cooperation with the University Hospital Pharmacy LMU. Results: 43 of the 47 included breast cancer patients (91%) used BB-CAM at some point during their treatment period. We found a significant increase from time point 1 (n = 27) to time point 2 (n = 40) (p = 0.004). Moreover, in the subgroup of newly diagnosed patients, the number significant rose from 17 at time point 1 to 28 at time point 2 (p = 0.007). Overall, we found potentially clinically relevant interactions in 30 of 43 patients (70%). Sixty interactions were detected at both times of investigations. Twenty-three different kinds of BB-CAM-to-BB-CAM (time point 1 [n = 12], time point 2 [n = 11]) or conventional medicine-to-BB-CAM interactions (time point 1 [n = 15], time point 2 [n = 22]) were discovered. Importantly, there was not a single interaction between BB-CAM and an anticancer drug. Conclusion: Breast cancer patients frequently use BB-CAM. Interactions were detected at both time points of investigation (time point 1 [n = 27], time point 2 [n = 33]). Interactions were particularly evident between BB-CAM substances as well as between BB-CAM and the patients' medication for pre-existing illnesses. Although no interaction between BB-CAM and an anticancer therapy was found, the use of BB-CAM should be evaluated at the beginning and regularly during therapy in view of the substantial number of interactions detected and the large number of upcoming targeted therapies.

Subsensory stochastic electrical stimulation targeting muscle afferents alters gait control during locomotor adaptations to haptic perturbations.

Subsensory noise stimulation targeting sensory receptors has been shown to improve balance control in healthy and impaired individuals. However, the potential for application of this technique in other contexts is still unknown. Gait control and adaptation rely heavily on the input from proprioceptive organs in the muscles and joints. Here we investigated the use of subsensory noise stimulation as a means to influence motor control by altering proprioception during locomotor adaptations to forces delivered by a robot. The forces increase step length unilaterally and trigger an adaptive response that restores the original symmetry. Healthy participants performed two adaptation experiments, one with stimulation applied to the hamstring muscles and one without. We found that participants adapted faster but to a lesser extent when undergoing stimulation. We argue that this behavior is because of the dual effect that the stimulation has on the afferents encoding position and velocity in the muscle spindles.

App-Based Lifestyle Coaching (PINK!) Accompanying Breast Cancer Patients and Survivors to Reduce Psychological Distress and Fatigue and Improve Physical Activity: A Feasibility Pilot Study.

Introduction: This pilot study aimed to investigate the effects of using an app-based certified medical product named PINK! on breast cancer patients and survivors. The objectives were to measure psychological distress, physical activity, and therapy-related fatigue of patients using PINK! to identify trends and develop a study design for a subsequent multicentric proof of efficacy RCT. Materials and Methods: PINK! offers individualized, evidence-based therapy and side-effect management, mindfulness-based stress reduction, nutritional and psychological education, physical activity tracking, and motivational exercises to implement lifestyle changes sustainably in daily routine. A prospective, intraindividual RCT was performed with n = 60 patients in 2021 at Comprehensive Cancer Center Munich. Patients with BC were included independent of the stage of diseases. The intervention group got access to PINK! over 12 weeks. Control group served as a waiting-list comparison to "standard of care." Results: Primary efficacy variable analysis revealed a relative average decrease of 32.9% in psychological distress, which corresponds to a statistically significant reduction (p < 0.001) within 12 weeks compared to the control group. Linear regressions within usage groups showed a correlation of high app usage and a reduction of psychological distress. Fatigue data presented a statistically significant antifatigue efficacy (p < 0.001) and physical activity increased by 63.9%. Conclusion: App-based supportive care offers a promising, low-threshold, and cost-efficient opportunity to improve psychological well-being, quality of life, fatigue, and physical activity. More research is needed to implement eHealth solutions in clinical cancer care.

Health-related quality of life and patient-centred outcomes with COVID-19 vaccination in patients with breast cancer and gynaecological malignancies.

Introduction: Safety and tolerability of COVID-19 vaccines were demonstrated by several clinical trials which led to the first FDA/EMA approvals in 2021. Because of mass immunizations, most social restrictions were waived with effects on quality of life. Therefore, our a-priori hypothesis was that COVID-19 vaccination impacted the health-related quality of life (HR-QoL) in patients with breast and gynecological cancer. Methods: From March 15th until August 11th, 2022, fully vaccinated patients with breast and gynecological cancer treated in the oncological outpatient clinics of the Department of Obstetrics and Gynecology, LMU University Hospital, Munich, Germany filled out a vaccine related QoL survey. Patients were asked about demographics (age, comorbidities), clinical parameters related to previous COVID-19 infections, and HR-QoL related parameters (living situation, responsibilities in everyday life). Subsequently, a questionnaire with 12 items was designed using a 5-point Likert scale (0 - strongly disagree/4 - strongly agree), covering the aspects health and therapy, social environment, participation in everyday life and overall assessment. Results: By August 11th, 2022, 108 out of 114 (94.7%) patients had received at least three doses of COVID-19 vaccine and six patients at least two doses. More than half of the surveyed patients were >55y (52.6%; mean: 55.1y, range 29-86y). Patients with breast cancer (n= 83) had early (59.0%) or metastatic cancer (41.0%); gynecological cancers (n=31) also included metastatic (54.8%) and non-metastatic cancer (45.2%). 83.3% of the patients stated that COVID-19 vaccination had a positive impact on their HR-QoL. Furthermore, 29 patients (25.4%) had undergone a COVID-19 infection. These patients reported self-limiting symptoms for a median duration of 5.9 days and no hospital admissions were registered. Conclusions: Our study demonstrates that vaccination against COVID-19 was positively associated with HR-QoL in patients with breast and gynecological cancer. Furthermore, vaccinated patients who underwent COVID-19 disease experienced only self-limiting symptoms.

Depth of response of induction therapy and consecutive maintenance treatment in patients with RAS wild-type metastatic colorectal cancer: An analysis of the PanaMa trial (AIO KRK 0212).

BACKGROUND: In patients with RAS wild-type metastatic colorectal cancer, depth of response (DpR) has gained importance as a novel end-point in clinical trials. We investigated the overall DpR, as well as the prognostic and predictive impact of DpR to induction therapy (six cycles of 5-fluorouracil, leucovorin [FU/FA], oxaliplatin [FOLFOX] and panitumumab [Pmab]) on consecutive maintenance therapy (FU/FA plus Pmab or FU/FA alone) in patients treated within the PanaMa trial. METHODS: Central radiological assessment was performed according to RECIST 1.1. DpR was defined as percentage change in tumour diameter within defined time intervals (induction therapy, maintenance therapy, total course of therapy). For prognostic and predictive analyses, median DpR (

An RNA-based system to study hepatitis B virus replication and evaluate antivirals.

Hepatitis B virus (HBV) chronically infects an estimated 300 million people, and standard treatments are rarely curative. Infection increases the risk of liver cirrhosis and hepatocellular carcinoma, and consequently, nearly 1 million people die each year from chronic hepatitis B. Tools and approaches that bring insights into HBV biology and facilitate the discovery and evaluation of antiviral drugs are in demand. Here, we describe a method to initiate the replication of HBV, a DNA virus, using synthetic RNA. This approach eliminates contaminating background signals from input virus or plasmid DNA that plagues existing systems and can be used to study multiple stages of HBV replication. We further demonstrate that this method can be uniquely applied to identify sequence variants that confer resistance to antiviral drugs.

High cell surface death receptor expression determines type I versus type II signaling.

Previous studies have suggested that there are two signaling pathways leading from ligation of the Fas receptor to induction of apoptosis. Type I signaling involves Fas ligand-induced recruitment of large amounts of FADD (FAS-associated death domain protein) and procaspase 8, leading to direct activation of caspase 3, whereas type II signaling involves Bid-mediated mitochondrial perturbation to amplify a more modest death receptor-initiated signal. The biochemical basis for this dichotomy has previously been unclear. Here we show that type I cells have a longer half-life for Fas message and express higher amounts of cell surface Fas, explaining the increased recruitment of FADD and subsequent signaling. Moreover, we demonstrate that cells with type II Fas signaling (Jurkat or HCT-15) can signal through a type I pathway upon forced receptor overexpression and that shRNA-mediated Fas down-regulation converts cells with type I signaling (A498) to type II signaling. Importantly, the same cells can exhibit type I signaling for Fas and type II signaling for TRAIL (TNF-α-related apoptosis-inducing ligand), indicating that the choice of signaling pathway is related to the specific receptor, not some other cellular feature. Additional experiments revealed that up-regulation of cell surface death receptor 5 levels by treatment with 7-ethyl-10-hydroxy-camptothecin converted TRAIL signaling in HCT116 cells from type II to type I. Collectively, these results suggest that the type I/type II dichotomy reflects differences in cell surface death receptor expression.